The ESI-MS spectra of PRWT and PRD25N revealed four peaks of differently charged ions in the range of mass/charge ratio (m/z) of 1,500-2,500. Because +5 charged monomer ion and +10 charged dimer ion have the same m/z (m/z =2,164.77 as calculated with their average mass in the case of PRD25N), the greatest peak detected at m/z 2,164.51 was determined to represent two forms, a PR monomer and PR dimer, thus being [PRD25N]5+ and [2PRD25N]10+. In the present report hereafter, we designate a monomer and a dimer ion of PRX as [PRX]Y and [2PRX]Y, respectively, where X denotes an amino acid substitution(s) and Y denotes a charge of ion. To determine whether the detected ions represented monomers and/or dimers, we examined multiply charged isotopologue clusters of PRD25N using the Solarix FT-ICR MS (Bruker Daltonics) and analyzed the difference in m/z ratios of two adjacent isotope peaks (delta m/z) because monomer and dimer PRD25N ions with the same m/z show different delta m/z values in order of their charges. We also constructed three mutated PR species containing amino acid substitutions at the active site (PRD29N, PRT26A, and PRR87K), a C terminus-truncated mutant (PR1-C95A), and a PR carrying L97A and F99A substitutions (PR97/99). In Figure 2C, two PRD29N dimer ions ([2PRD29N]11+ and [2PRD29N]9+) were detected, whereas no dimer ions were detected with PRT26A and PRR87K. Additional analyses of the isotopologue ion peaks with PRT26A and PRR87K confirmed the absence of dimer ions. Importantly, two PR1-C95A dimer ions ([2PR1-C95A]11+ and [2PR1-C95A]9+) were identified, although PR1-C95A monomer ion ([PR1-C95A]6+) was found to be a major peak. Two dimer ions were also detected in the case of PR97/99, as shown in Figure 3C. Considering that [PRWT]5++[2PRWT]10+ representing monomers+dimers was found to be a major peak together with a minor peak of [PRWT]6+ in Figure 2A, the PR1-C95A and PR97/99 species were thought to have a significantly reduced but persistent ability to dimerize in comparison with PRWT. Taken together, the data strongly suggest that the PR dimerization process consists of two distinct steps: (i) initial albeit weak intermolecular interactions occurring in the active site interface, constructing unstable or transient dimers, and (ii) subsequent interactions in the termini interface, resulting in the complete and tighter PR dimerization. In an attempt to examine the thermal stability of PRWT and various mutated PR species mentioned above, we conducted DSF. As illustrated in Figure 4, the order of thermal stability was PRWT PRD25N PRD29N PR32/33/54/84 PR97/99, PRT26A PRR87K PR1-C95A (Tm; 53.37 52.18 51.02 50.17 48.22, 48.12 47.02 44.46 C, respectively). The difference in Tm values (deltaTm) between PRD25N and PRD29N (1.16 C) was less than deltaTm between PRD25N and PR1-C95A (7.72 C), indicating that in terms of thermal stability, PRD25N is closer to PRD29N compared with the most unstable PR1-C95A. Thus, PRD29N monomer subunits are likely to interact at the active site interface and subsequently, at the termini interface, forming stable dimers. The DSF data, however, showed that Tm value of PR97/99 (48.22 C) was quite low compared with that of PRWT (53.37 C) and PRD29N (51.02 C), suggesting that PR97/99 dimers are likely to be unstable. The Tm value of PR1-C95A was further lower (44.46 C), suggesting that PR1-C95A dimers are also likely to be unstable. Taking the ESI-MS and DSF results together, one can presume that the present ESI-MS assay detects both unstable (transient) and stable dimers. Furthermore, the present DSF data indicate that the stability of PR97/99 and PR1-C95A dimers was lower than that of PRD25N dimer (dimer dissociation constant; KD = 1.3 microM), suggesting that the KD values of transient dimers were higher than 1.3 microM. Thus, the present DSF data corroborate the above ESI-MS data showing that the HIV-1 PR dimerization process undergoes two steps. It is worth noting that we have most recently found that GRL-0142 binds significantly more to monomers than to dimers compared with DRV. We have also found that a mutated protease, a transframe precursor form of PR containing a D25N substitution (TFR-PRD25N), which does not dimerize and is well bound by GRL-0142. We are currently trying to crystalize GRL-0142-bound TFR-PRD25N using various crystallization conditions.